Metal ions play a crucial role in a myriad of biological processes, and the ability to monitor real time changes in metal ion concentrations is essential for understanding a variety of physiological events. Alterations in cellular homeostasis of metal ions are connected to human disorders and diseases including cancer, diabetes, and neurodegenerative disease (Nat. Chem. Biol., 2008; 4: 168-175). As demonstrated in the cartoon of FIG. 1, metal ion signaling and homeostasis play an important role in many cellular processes. The main metal ions involved are the monovalent sodium and potassium, and the bivalent calcium, zinc magnesium and iron. Cells maintain healthy levels of the essential metal ions using several classes of proteins. Currently, imaging dynamic changes in metal ions levels is restricted to fluorescence-based methodologies which are limited by low tissue penetration and thus do not allow in vivo imaging of metal ions in deep tissues or organs.
Chemical exchange saturation transfer (CEST) MR imaging is a technique in which low-concentration marker molecules are labeled by saturating their exchangeable protons (e.g., hydroxyl, amine, amide, or imino protons) by radio-frequency (RF) irradiation. If such saturation can be achieved rapidly (i.e., before the proton exchanges), exchange of such labeled protons with water leads to progressive water saturation, allowing indirect detection of the solute via the water resonance through a decrease in signal intensity in MRI [Ward, K. M., Aletras, A. H. & Balaban, R. S. A new class of contrast agents for MRI based on proton chemical exchange dependent saturation transfer (CEST). J Magn Reson 143, 79-87 (2000)].
Each CEST contrast agent can have a different saturation frequency, which depends on the chemical shift of the exchangeable proton. The magnitude of proton transfer enhancement (PTE) due to this effect, and the resulting signal reduction from equilibrium (S0) to saturated (S), are given by [Goffeney, N., Bulte, J. W., Duyn, J., Bryant, L. H., Jr. & van Zijl, P. C. Sensitive NMR detection of cationic-polymer-based gene delivery systems using saturation transfer via proton exchange. J Am Chem Soc 123, 8628-8629 (2001)]:
                              PTE          =                                                                      NM                  w                                ⁢                α                ⁢                                                                  ⁢                                  k                  ex                                                                                                  (                                          1                      -                                              x                        CA                                                              )                                    ⁢                                      R                                          1                      ⁢                                                                                          ⁢                      wat                                                                      +                                                      x                    CA                                    ⁢                                      k                    ex                                                                        ·                          {                              1                -                                  e                                                            -                                              [                                                                                                            (                                                              1                                -                                                                  x                                  CA                                                                                            )                                                        ⁢                                                          R                                                              1                                ⁢                                                                                                                                  ⁢                                wat                                                                                                              +                                                                                    x                              CA                                                        ⁢                                                          k                              ex                                                                                                      ]                                                              ⁢                                          t                      sat                                                                                  }                                      ,                            [                  Eq          .                                          ⁢          1                ]                                          and          ⁢                                          ⁢                      (                          1              -                                                S                  sat                                /                                  S                  0                                                      )                          =                                            PTE              ·                              [                CA                ]                                                    2              ·                              [                                                      H                    2                                    ⁢                  O                                ]                                              .                                    [                  Eq          .                                          ⁢          2                ]            “CA” is the contrast agent containing multiple exchangeable protons, xCA its fractional exchangeable proton concentration, a the saturation efficiency, k the pseudo first-order rate constant, N the number of exchangeable protons per molecular weight unit, and Mw the molecular weight of the CA. The exponential term describes the effect of back exchange and water longitudinal relaxation (R1wat=1/T1wat) on the transfer during the RF saturation period (tsat). This effect will be bigger when protons exchange faster, but the catch is that saturation occurs faster as well, which increases the radio-frequency power needed. In addition, the resonance of the particular protons must be well separated from water in the proton NMR spectrum, which requires a slow exchange on the NMR time scale. This condition means that the frequency difference of the exchangeable protons with water is much larger than the exchange rate (Δω>k).
Thus, the CEST technology becomes more applicable at higher magnetic fields or when using paramagnetic shift agents [Mang, S., Merritt, M., Woessner, D. E., Lenkinski, R. E. & Sherry, A. D. PARACEST agents: modulating MRI contrast via water proton exchange. Acc Chem Res 36, 783-790 (2003)]. Any molecule that exhibits a significant PTE effect can be classified as a CEST (contrast) agent. The concept of these agents as MR contrast agents is somewhat similar to the chemical amplification of colorimetric labels in in situ gene expression assays. CEST agents can be detected by monitoring the water intensity as a function of the saturation frequency, leading to a so-called z-spectrum. In such spectra, the saturation effect of the contrast agent on the water resonance is displayed as a function of irradiation frequency.
Since the first report of CEST contrast in 2000, CEST MR imaging has become widely used MRI contrast mechanism (demonstrated in FIG. 2). FIG. 2 shows that a CEST contrast is generated by the dynamic exchange process between an exchangeable proton of a biomarker of interest and the surrounding water protons. To detect the biomarkers, the magnetization of some of their exchangeable protons is nullified by applying a selective radiofrequency saturation pulse at the specific resonance frequency (chemical shift) of the target protons. Due to exchange of the “saturated” agent protons with surrounding water protons, the net water signal is reduced thus enhancing the MRI contrast.
CEST-MRI has been employed for many applications in molecular and cellular MRI (see, e.g., Bar-Shir, A. et al., J. Am Chem. Soc. 2013; Ratnakar, S. J. et al., J. Am Chem. Soc. 2012, 134, 5798; Liu, G. et al., Magn Reson Med. 2012, 67, 1106; Longo, D. L. et al., Magn Reson Med. 2012, doi: 10.1002/mrm. 24513; Li., Y. et al. Contrast Media Mol Imaging 2011, 6, 219; Aime, S. et al. Angew Chem Int Ed Engl 2005, 44, 1813; Chan, K. W. et al., Nat Mat 2013, 12, 268; Liu, G. et al., NMR in Biomedicine 2013, doi: 10.1002/nbm.2899).
However, despite that recent advances in the field of molecular magnetic resonance imaging (MRI) has led to the development of new strategies in the design and synthesis of responsive MRI contrast agents for detecting biologically relevant metal ions, the specificity and sensitivity of those probes is limited.
Therefore, there is a need for the development of novel methodology and responsive MRI contrast agents for detecting biologically relevant metal ions with improved specificity and sensitivity.